Cyclic Adenosine Monophosphate, Inositol 1,4,5-trisphosphate, Calcium, and Phosphorylated Myosin Light Chain Regulation Through M2 and M3 Muscarinic Receptors of Scleral Fibroblast Cells in Rat Myopia Model

Authors

  • Nanda Wahyu Anandita Doctoral Program of Medical Science, Faculty of Medicine, Universitas Brawijaya, Malang, East Java, Indonesia; Department of Ophthalmology, Faculty of Medicine, Universitas Brawijaya, Dr. Saiful Anwar Hospital, Malang, East Java, Indonesia
  • Nurdiana Nurdiana Department of Pharmacology, Faculty of Medicine, Universitas Brawijaya, Malang, East Java, Indonesia
  • Endang Sri Wahyuni Department of Physiology, Faculty of Medicine, Universitas Brawijaya, Malang, East Java, Indonesia
  • Hidayat Sujuti Department of Ophthalmology, Faculty of Medicine, Universitas Brawijaya, Dr. Saiful Anwar Hospital, Malang, East Java, Indonesia

DOI:

https://doi.org/10.3889/oamjms.2021.6752

Keywords:

Phosphorylated Myosin Light Chain, Myopia, Sclera, Himbacine, Methoctramine

Abstract

AIM: This study aims to investigate the concentration of cyclic adenosine monophosphate (cAMP), inositol 1,4,5-trisphosphate (IP3), calcium (Ca2+), and the expression phosphorylated myosin light chain (MLC) in Rattus norvegicus scleral fibroblast cells.

METHODOLOGY: This study utilized an in vitro experimental study by applying Rattus norvegicus scleral fibroblast cell culture. The cultured cells were divided into control and lens-induced myopia (LIM) groups. The control and LIM culture groups were each divided into five groups, namely, negative control, 0.1 μM acetylcholine, 0.1 μM himbacine, 0.1 μM methoctramine, and 0.1 μM 4-DAMP group. The cAMP, IP3, and Ca2+ concentration were analyzed in the 0th, 5th, 10th, 20th, and 30th. The phosphorylated MLC expression was analyzed using confocal microscope.

RESULTS: In the LIM group, the highest cAMP concentration is visible at the 10th min on the himbacine group (0.304 ± 0; p = 0.043) and on the 4-DAMP group (0.346 ± 0; p = 0.043). The highest IP3 concentration is found on the LIM group at the 20th min in comparison to the control group (2503.6 ± 11 vs. 2039.2 ± 2.1; p = 0.046). The highest Ca2+ concentration belongs to the 4-DAMP treatment group from the 5th to the 30th min. The highest average phosphorylated MLC expression value in the LIM group is shown by the 0.1μM 4-DAMP treatment (184.2 ± 37.9c au).

CONCLUSION: The regulation of cAMP, IP3, Ca2+, and phosphorylated MLC on the M2 and M3 muscarinic receptor of the scleral fibroblast cells of myopia animal models differs from normal animal models which may be due to interactions of M2 and M3 muscarinic receptor as compensation reaction or crosstalk on myopia induction.

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References

Metlapally R, Wildsoet CF. Scleral mechanisms underlying ocular growth and myopia. In: Progress in Molecular Biology and Translational Science; 2015. https://doi.org/10.1016/bs.pmbts.2015.05.005 DOI: https://doi.org/10.1016/bs.pmbts.2015.05.005

Shinohara K, Yoshida T, Liu H, Ichinose S, Ishida T, Nakahama KI, et al. Establishment of novel therapy to reduce progression of myopia in rats with experimental myopia by fibroblast transplantation on sclera. J Tissue Eng Regen Med. 2018;12(1):e451-61. https://doi.org/10.1002/term.2275 PMid:28401697 DOI: https://doi.org/10.1002/term.2275

Jiang X, Kurihara T, Kunimi H, Miyauchi M, Ikeda SI, Mori K, et al. A highly efficient murine model of experimental myopia. Sci Rep. 2018;8(1):2026. https://doi.org/10.1038/s41598-018-20272-w PMid:29391484 DOI: https://doi.org/10.1038/s41598-018-20272-w

Tao Y, Pan M, Liu S, Fang F, Lu R, Lu C, et al. cAMP level modulates scleral collagen remodeling, a critical step in the development of myopia. PLoS One. 2013;8(8):e71441. https://doi.org/10.1371/journal.pone.0071441 PMid:23951163 DOI: https://doi.org/10.1371/journal.pone.0071441

Srinivasalu N, Lu C, Pan M, Reinach PS, Wen Y, Hu Y, et al. Role of cyclic adenosine monophosphate in myopic scleral remodeling in Guinea pigs: A microarray analysis. Invest Ophthalmol Vis Sci. 2018;59(10):4318-25. https://doi.org/10.1167/iovs.17-224685 PMid:30167661 DOI: https://doi.org/10.1167/iovs.17-224685

Halls ML, Cooper DM. Regulation by Ca2+-signaling pathways of adenylyl cyclases. Cold Spring Harb Perspect Biol. 2011;3(1):a004143. https://doi.org/10.1101/cshperspect. a004143 PMid:21123395 DOI: https://doi.org/10.1101/cshperspect.a004143

Ockenga W, Kühne S, Bocksberger S, Banning A, Tikkanen R. Non-neuronal functions of the M2 muscarinic acetylcholine receptor. Genes. 2013;4(2):171-97. https://doi.org/10.3390/genes4020171 PMid:24705159 DOI: https://doi.org/10.3390/genes4020171

Tanzarella P, Ferretta A, Barile S, Ancona M, De Rasmo D, Signorile A, et al. Increased levels of cAMP by the calcium-dependent activation of soluble adenylyl cyclase in parkin-mutant fibroblasts. Cells. 2019;8(3):250. https://doi.org/10.3390/cells8030250 PMid:30875974 DOI: https://doi.org/10.3390/cells8030250

Berridge MJ. The inositol trisphosphate/calcium signaling pathway in health and disease. Physiol Rev. 2016;96(4):1261- 96. https://doi.org/10.1152/physrev.00006.2016 PMid:27512009 DOI: https://doi.org/10.1152/physrev.00006.2016

Taylor CW. Regulation of IP3 receptors by cyclic AMP. Cell Calcium. 2017;63:48-52. https://doi.org/10.1016/j.ceca.2016.10.005 PMid:27836216 DOI: https://doi.org/10.1016/j.ceca.2016.10.005

Prole DL, Taylor CW. Inositol 1, 4, 5-trisphosphate receptors and their protein partners as signalling hubs. J Physiol. 2016;594(11):2849-66. https://doi.org/10.1113/jp271139 PMid:26830355 DOI: https://doi.org/10.1113/JP271139

Khapchaev AY, Shirinsky VP. Myosin light chain kinase MYLK1: Anatomy, interactions, functions, and regulation. Biochemistry (Mosc). 2016;81(13):1676-97. https://doi.org/10.1134/ s000629791613006x PMid:28260490 DOI: https://doi.org/10.1134/S000629791613006X

Kolodney MS, Elson EL. Correlation of myosin light chain phosphorylation with isometric contraction of fibroblasts. J Biol Chem. 1993;268(32):23850-5. https://doi.org/10.1016/s0021-9258(20)80463-3 PMid:8226923 DOI: https://doi.org/10.1016/S0021-9258(20)80463-3

McBrien NA, Jobling AI, Gentle A. Biomechanics of the sclera in myopia: Extracellular and cellular factors. Optom Vis Sci. 2009;86(1):E23-30. https://doi.org/10.1097/ opx.0b013e3181940669 PMid:19104466 DOI: https://doi.org/10.1097/OPX.0b013e3181940669

Jobling AI, Gentle A, Metlapally R, McGowan BJ, McBrien NA. Regulation of scleral cell contraction by transforming growth factor-β _and stress: Competing roles in myopic eye growth. J Biol Chem. 2009;284(4):2072-9. https://doi.org/10.1074/jbc.m807521200 PMid:19011237 DOI: https://doi.org/10.1074/jbc.M807521200

Andreollo NA, dos Santos EF, Araújo MR, Lopes LR. Rat’s age versus human’s age: What is the relationship? Arq Bras Cir Dig. 2012;25(1):49-51. PMid:22569979 DOI: https://doi.org/10.1590/S0102-67202012000100011

Zhou X, Pardue MT, Iuvone PM, Qu J. Dopamine signaling and myopia development: What are the key challenges. Prog Retin Eye Res. 2017;61:60-71. https://doi.org/10.1016/j.preteyeres.2017.06.003 PMid:28602573 DOI: https://doi.org/10.1016/j.preteyeres.2017.06.003

Xiao H, Fan ZY, Tian XD, Xu YC. Comparison of form-deprived myopia and lens-induced myopia in guinea pigs. Int J Ophthalmol. 2014;7(2):245-50. PMid:24790865

Mao J, Liu S, Fu C. Citicoline retards myopia progression following form deprivation in guinea pigs. Exp Biol Med (Maywood). 2016;241(11):1258-63. https://doi.org/10.1177/1535370216638773 PMid:26979720 DOI: https://doi.org/10.1177/1535370216638773

Wang S, Liu S, Mao J, Wen D. Effect of retinoic acid on the tight junctions of the retinal pigment epithelium-choroid complex of guinea pigs with lens-induced myopia in vivo. Int J Mol Med. 2014;33(4):825-32. https://doi.org/10.3892/ijmm.2014.1651 PMid:24535401 DOI: https://doi.org/10.3892/ijmm.2014.1651

Anandita NW, Sujuti H, Wahyuni ES, Nurdiana N. Binding affinity of selective inhibitors for M2 and M3 muscarinic acetylcholine receptors-in silico study for controlling myopia progressivity. Drug Invent Today. 2019;12(11):2624-8.

Barathi VA, Kwan JL, Tan QS, Weon SR, Seet LF, Goh LK, et al. Muscarinic cholinergic receptor (M2) plays a crucial role in the development of myopia in mice. Dis Model Mech. 2013;6(5):1146-58. https://doi.org/10.1242/dmm.010967 PMid:23649821 DOI: https://doi.org/10.1242/dmm.010967

Unno T, Matsuyama H, Sakamoto T, Uchiyama M, Izumi Y, Okamoto H, et al. M(2) and M(3) muscarinic receptor-mediated contractions in longitudinal smooth muscle of the ileum studied with receptor knockout mice. Br J Pharmacol. 2005;146(1):98-108. https://doi.org/10.1038/sj.bjp.0706300 PMid:15965495 DOI: https://doi.org/10.1038/sj.bjp.0706300

Resende RR, Adhikari A. Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation. Cell Commun Signal. 2009;7:20. https://doi.org/10.1186/1478-811x-7-20 PMid:19712465 DOI: https://doi.org/10.1186/1478-811X-7-20

Prole DL, Taylor CW. Structure and function of ip3 receptors. Cold Spring Harb Perspect Biol. 2019;11(4):a035063. https://doi.org/10.1101/cshperspect.a035063 PMid:30745293 DOI: https://doi.org/10.1101/cshperspect.a035063

Lincoln TM. Myosin phosphatase regulatory pathways: Different functions or redundant functions? Circ Res. 2007;100(1):10-2. https://doi.org/10.1161/01.res.0000255894.25293.82 PMid:17204659 DOI: https://doi.org/10.1161/01.RES.0000255894.25293.82

Hirano K, Derkach DN, Hirano M, Nishimura J, Kanaide H. Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain. Mol Cell Biochem. 2003;248(1-2):105-14. PMid:12870661

Unno T, Matsuyama H, Izumi Y, Yamada M, Wess J, Komori S. Roles of M2 and M3 muscarinic receptors in cholinergic nerve-induced contractions in mouse ileum studied with receptor knockout mice. Br J Pharmacol. 2006;149(8):1022-30. https://doi.org/10.1038/sj.bjp.0706955 PMid:17099717 DOI: https://doi.org/10.1038/sj.bjp.0706955

Hornigold DC, Mistry R, Raymond PD, Blank JL, Challiss RA. Evidence for cross-talk between M2 and M3 muscarinic acetylcholine receptors in the regulation of second messenger and extracellular signal-regulated kinase signalling pathways in Chinese hamster ovary cells. Br J Pharmacol. 2003;138(7):1340-50. https://doi.org/10.1038/sj.bjp.0705178 PMid:12711635 DOI: https://doi.org/10.1038/sj.bjp.0705178

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Published

2021-08-26

How to Cite

1.
Anandita NW, Nurdiana N, Wahyuni ES, Sujuti H. Cyclic Adenosine Monophosphate, Inositol 1,4,5-trisphosphate, Calcium, and Phosphorylated Myosin Light Chain Regulation Through M2 and M3 Muscarinic Receptors of Scleral Fibroblast Cells in Rat Myopia Model. Open Access Maced J Med Sci [Internet]. 2021 Aug. 26 [cited 2024 Mar. 29];9(B):890-9. Available from: https://oamjms.eu/index.php/mjms/article/view/6752

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